Process for the production of expression vectors comprising at least one stochastic sequence of polynucleotides

Abstract

The present invention is directed to a process for the production of a peptide, polypeptide, or protein having a predetermined property. In accordance with one embodiment, the process begins by producing by way of synthetic polynucleotide coupling, stochastically generated polynucleotide sequences. A library of expression vectors containing such stochastically generated polynucleotide sequences is formed. Next, host cells containing the vectors are cultured so as to produce peptides, polypeptides, or proteins encoded by the stochastically generated polynucleotide sequences. Screening or selection is carried out on such host cells to identify a peptide, polypeptide, or protein produced by the host cells which has the predetermined property. The stochastically generated polynucleotide sequence which encodes the identified peptide, polypeptide, or protein is then isolated and used to produce the peptide, polypeptide, or protein having the predetermined property.

We claim: 
     
       1. A process for the production of an expression vector which comprises at least one stochastic sequence of polynucleotides, comprising the steps of: providing in an appropriate buffer at least three different sequences of oligonucleotides, said oligonucleotides each comprising at least 7 nucleotide residues;   polymerizing said oligonucleotides to form a stochastic sequence of polynucleotides; and   ligating said stochastic sequence of polynucleotides into a linearized expression vector.   
     
     
       2. The process according to claim 1 wherein the oligonucleotides further comprise a heptamer. 
     
     
       3. The process according to claim 1, said process further comprising the steps of: transforming a competent clone with said ligated expression vector;   culturing said transformed competent clone;   purifying said expression vector from said amplified competent clone;   isolating said cultured stochastic sequence of polynucleotides from said expression vector;   cutting said stochastic sequence of polynucleotides by means of at least one restriction enzyme which corresponds to a specific restriction enzyme site present in said stochastic sequence of polynucleotides;   treating said cut stochastic sequence of polynucleotides with a T4 ligase to create a new ensemble of stochastic sequence of polynucleotides containing a new stochastic sequence of polynucleotides; and   ligating said new ensemble of stochastic sequence of polynucleotides into an expression vector.   
     
     
       4. The process according to claim 2 wherein the heptamers are palindromic. 
     
     
       5. The process according to claim 4 wherein the palindromic heptamers are selected from the group consisting of: 5' XTCGCGA 3';   5' XCTGCAG 3'; and   5' RGGTACC 3'; where X=A, G, C, or T, and R=A or T.     
     
     
       6. The process according to claim 1 wherein the oligonucleotides further comprise an octomer. 
     
     
       7. The process according to claims 1 or 6, said process further comprising the steps of: transforming a competent clone with said ligated expression vector;   culturing said transformed competent clone;   purifying and isolating said expression vector from said cultured competent clone;   cutting said stochastic sequence of polynucleotides by means of at least one restriction enzyme which corresponds to said specific restriction enzyme site present in said stochastic sequence of polynucleotides;   treating said cut stochastic sequence of polynucleotides with a T4 ligase to create a new ensemble of stochastic sequence of polynucleotides comprising a new stochastic sequence of polynucleotides; and   ligating said new ensemble of stochastic sequence of polynucleotides into an expression vector.   
     
     
       8. The process according to claim 6 wherein the octamers are palindromic. 
     
     
       9. The process according to claim 8 wherein the palindromic octamers are selected from the group consisting of: 5' GGAATTCC 3';   5' GGTCGACC 3';   5' CAAGCTTG 3';   5' CCATATGG 3'; and   5' CATCGATG 3'.   
     
     
       10. The process according to claim 1 wherein the stochastic double stranded DNA fragments are about 160 to 800 base pairs in length. 
     
     
       11. A process for the production of an expression vector capable of producing a transcription product or a translation product comprising at least one stochastic sequence of polynucleotides, comprising the steps of: linearizing an expression vector;   reacting said linearized expression vector with terminal transferase enzyme in the presence of desired ratios of deoxynucleotide-triphosphates of guanine, cytosine, thymidine, and adenine to form a stochastic polynucleotide sequence at each 3' extremity of said linearized vector;   hybridizing said stochastic polynucleotide sequence at a 3' extremity of said linearized vector; and   synthesizing a second strand from said 3' ends of said hybridized vector by incubating with polymerase.   
     
     
       12. A process for the production of a library of expression vectors capable of producing a transcription product or a translation product, said vectors comprising at least one stochastic sequence of polynucleotides, comprising the steps of: producing at least one stochastic sequence of polynucleotides;   ligating said stochastic sequence of polynucleotides into an expression vector;   transforming a competent clone with said ligated expression vector;   culturing said transformed clone;   screening and/or selecting said transformed clone in order to isolate a clone expressing a stochastic polynucleotide leading to the synthesis of a transcription product or a translation product;   isolating said selected or screened transformed clone; and   isolating the expression vector cultured in said selected or screened transformed clone so identified.   
     
     
       13. A library of expression vectors capable of producing a transcription product or a translation product, said expression vectors comprising at least one stochastic sequence of polynucleotides, produced in accordance with the process of claim 12. 
     
     
       14. An expression vector produced in accordance with the process of claim 1, 11, 12. 
     
     
       15. The process according to claims 11 or 12 wherein said translation product comprises a product having a desired property and is selected from the group consisting of a peptide, a polypeptide or a protein. 
     
     
       16. The process according to claims 1 or 12 wherein said transcription product comprises a product having a desired property and is selected from the group consisting of a RNA or a DNA. 
     
     
       17. The library of expression vectors according to claim 13 wherein said library comprises stochastic nucleotide sequences encoding for at least 10,000 peptides, polypeptides or proteins. 
     
     
       18. A method of producing a diverse population of vectors comprising: (a) synthesizing a diverse population of stochastically generated polynucleotide sequences comprising greater than about 1×10 5  different polynucleotide sequences, said method consisting of stochastic copolymerization of double stranded oligonucleotides, copolymerization of the four kinds of nucleotide triphosphates consisting of adenine, cytosine, guaninie and thymine, and chemical synthesis, and   (b) inserting said diverse population of stochastically generated polynucleotide sequences into a population of vectors to form a diverse population of vectors containing stochastically generated polynucleotide sequences.   
     
     
       19. The method of claim 18, which said diverse population of stochastically generated polynucleotide sequences, further comprise greater than about 1×10 6  different polynucleotide sequences. 
     
     
       20. The method of claim 18, which said diverse population of stochastically generated polynucleotide sequences, further comprise greater than about 1×10 7  different polynucleotide sequences. 
     
     
       21. The method of claim 18, wherein said diverse population of stochastically generated polynucleotide sequences, further comprise greater than about 1×10 8  different polynucleotide sequences. 
     
     
       22. The method of claim 18, wherein said inserting further comprises hybridization of complementary ends. 
     
     
       23. The method of claim 18, wherein said inserting further comprises ligation. 
     
     
       24. The method of claim 18, further comprising introducing said diverse population of vectors containing stochastically generated polynucleotide sequences into host cells. 
     
     
       25. The method of claim 18, wherein step (a) further comprises synthesizing a population of at least partially stochastic polynucleotide sequences. 
     
     
       26. The method of claim 18, wherein step (b) further comprises digesting the diverse population of vectors with a restriction enzyme having a recognition sequence absent in the expression vector and reinserting the digested products into said digested population of vectors to form a different population having a greater number of stochastic polynucleotide sequences. 
     
     
       27. A method of producing a diverse population of vectors, comprising stochastically copolymerizing a diverse population of vectors containing double stranded polynucleotides so as to produce a new population of vectors containing greater than about 1×10 5  different polynucleotide sequences. 
     
     
       28. The method of claim 27, wherein said new population of vectors further comprise greater than about 1×10 6  different polynucleotide sequences. 
     
     
       29. The method of claim 27, which said new population of vectors further comprise greater than about 1×10 7  different polynucleotide sequences. 
     
     
       30. The method of claim 27, wherein said new population of vectors further comprise greater than about 1×10 8  different polynucleotide sequences. 
     
     
       31. The method of claim 27, wherein said stochastic copolymerization is effected by hybridization of complementary ends. 
     
     
       32. The method of claim 27, wherein said stochastic copolymerization is effected by ligation. 
     
     
       33. The method of claim 27, further comprising introducing said diverse population of vectors containing stochastically generated polynucleotide sequences into host cells. 
     
     
       34. The method of claim 27, wherein said double stranded polynucleotides further comprise stochastic polynucleotide sequences. 
     
     
       35. The method of claim 27, wherein said double stranded polynucleotides further comprise at least partially stochastic polynucleotide sequences. 
     
     
       36. The method of claim 27, wherein said diverse population of vectors further comprises two or more diverse populations of vectors. 
     
     
       37. A method of producing a diverse populations of vectors, comprising: (a) obtaining one or more diverse populations of vectors containing diverse sequences of double stranded polynucleotides;   (b) digesting the one or more diverse populations of vectors with a restriction enzyme, and   (c) stochastically copolymerizing the one or more diverse populations of double stranded polynucleotides so as to produce a new population of greater than about 1×10 5  different polynucleotide sequences.   
     
     
       38. The method of claim 37, wherein said new population of vectors further comprise greater than about 1×10 6  different polynucleotide sequences. 
     
     
       39. The method of claim 37, wherein said new population of vectors further comprise greater than about 1×10 7  different polynucleotide sequences. 
     
     
       40. The method of claim 37, wherein said new population of vectors further comprise greater than about 1×10 8  different polynucleotide sequences. 
     
     
       41. The method of claim 37, wherein said stochastic copolymerization is effected by hybridization of complementary ends. 
     
     
       42. The method of claim 37, wherein said stochastic copolymerization is effected by ligation. 
     
     
       43. The method of claim 37, further comprising introducing said diverse population of vectors containing stochastically generated polynucleotide sequences into host cells. 
     
     
       44. The method of claim 37, wherein said double stranded polynucleotides further comprise stochastic polynucleotide sequences. 
     
     
       45. The method of claim 37, wherein said double stranded polynucleotides further comprise at least partially stochastic polynucleotide sequences. 
     
     
       46. The method of claim 37, wherein said diverse population of vectors further comprises two or more diverse populations of vectors.